The aim of the proposed research is to understand the catalytic mechanism of the Zn(II) metalloenzyme porphobilinogen synthase (also known as delta-aminolevulinate dehydratase, E.C. 4.2.1.24). Porphobilinogen (PBG) synthase catalyzes the asymmetic condensation of two molecules of 5-aminolevulinate (ALA) which is the second step in the biosynthesis of porphyrin (heme), chlorophyll, corrin (vitamin B12), cofactor F430 of the methanogenic bacteria, and a wide spectrum of tetrapyrrole pigments. PBG synthase activity is inhibited by the environmental toxin lead (Pb), which is believed responsible for the porphyria associated with Pb intoxication. Elucidation of the mechanism of Pb inhibition is of considerable clinical significance in view of the high levels of Pb which have accumulated in the biosphere. A variety of tools will be employed to dissect the chemical mechanism of PBG synthase and the mechanism of Pb inhibition. The substrate structure will be probed by 1H and 13C NMR and isotope exchange studies. The reaction intermediate structures will be probed by 13C and 15N NMR characterization of stoichiometric enzyme-substrate complexes. The demonstrated success of the 13C NMR approach to studying PBG synthase, a 280,000 dalton protein dramatically expands the use of this tool to study enzyme catalyzed reactions and promises to be of value to deciphering the mechanisms of other enzymes. The sequence of catalytic events and the nature of the transition states will be probed by isotope effect measurements. The environment of the Zn(II) ion will be probed spectroscopically. An X-ray crystallographic approach will be used to determine the three dimensional structure of PBG synthase. Methylmethanethiosulfonate modification of PBG synthase provides a method for preparing crystals with isomorphous metal ion replacement. Evaluation of the mechanism of Pb(II) inhibition will include NMR characterization of Pb(II) inhibited enzyme which is expected to identify the chemical step prior to Pb(II) inhibition. Identification of the perturbing factors which complicate the use of PBG synthase as a sensitive clinical diagnostic for Pb(II) intoxication will be continued. In summary, the proposed research utilizes a wide variety of techniques with the goal of dissecting 1) the chemical mechanism, including the role of Zn(II), 2) the mechanism of Pb(II) inhibition, and 3) the three dimensional structure of PBG synthase, an enzyme essential to all forms of life.